Mass Spectra and Yields of Intact Charged Biomolecules Ejected by Massive Cluster Impact for Bioimaging in a Time-of-Flight Secondary Ion Microscope.

Impacts of massive, highly charged glycerol clusters (≳10(6) Da, ≳ ± 100 charges) have been used to eject intact charged molecules of peptides, lipids, and small proteins from pure solid samples, enabling imaging using these ion species in a time-of-flight secondary ion microscope with few-micrometer spatial resolution. Here, we report mass spectra and useful ion yields (ratio of intact charged molecules detected to molecules sputtered) for several molecular species-two peptides, bradykinin and angiotensin II; two lipids, phosphatidylcholine and sphingomyelin; Irganox 1010 (a detergent); insulin; and rhodamine B-and show that useful ion yields are high enough to enable bioimaging of peptides and lipids in biological samples with few-micrometer resolution and acceptable signals. For example, several hundred molecular ion counts should be detectable from a 3 × 3 µm(2) area of a pure lipid bilayer given appropriate instrumentation or tens of counts from a minor constituent of such a layer.

Improving signal to noise in labeled biological specimens using energy-filtered TEM of sections with a drift correction strategy and a direct detection device.

Energy filtered transmission electron microscopy techniques are regularly used to build elemental maps of spatially distributed nanoparticles in materials and biological specimens. When working with thick biological sections, electron energy loss spectroscopy techniques involving core-loss electrons often require exposures exceeding several minutes to provide sufficient signal to noise. Image quality with these long exposures is often compromised by specimen drift, which results in blurring and reduced resolution. To mitigate drift artifacts, a series of short exposure images can be acquired, aligned, and merged to form a single image. For samples where the target elements have extremely low signal yields, the use of charge coupled device (CCD)-based detectors for this purpose can be problematic. At short acquisition times, the images produced by CCDs can be noisy and may contain fixed pattern artifacts that impact subsequent correlative alignment. Here we report on the use of direct electron detection devices (DDD's) to increase the signal to noise as compared with CCD's. A 3× improvement in signal is reported with a DDD versus a comparably formatted CCD, with equivalent dose on each detector. With the fast rolling-readout design of the DDD, the duty cycle provides a major benefit, as there is no dead time between successive frames.

Intracellular imaging of nanoparticles: is it an elemental mistake to believe what you see?

In order to understand how nanoparticles (NPs <100 nm) interact with cellular systems, potentially causing adverse effects, it is important to be able to detect and localize them within cells. Due to the small size of NPs, transmission electron microscopy (TEM) is an appropriate technique to use for visualizing NPs inside cells, since light microscopy fails to resolve them at a single particle level. However, the presence of other cellular and non-cellular nano-sized structures in TEM cell samples, which may resemble NPs in size, morphology and electron density, can obstruct the precise intracellular identification of NPs. Therefore, elemental analysis is recommended to confirm the presence of NPs inside the cell. The present study highlights the necessity to perform elemental analysis, specifically energy filtering TEM, to confirm intracellular NP localization using the example of quantum dots (QDs). Recently, QDs have gained increased attention due to their fluorescent characteristics, and possible applications for biomedical imaging have been suggested. Nevertheless, potential adverse effects cannot be excluded and some studies point to a correlation between intracellular particle localization and toxic effects. J774.A1 murine macrophage-like cells were exposed to NH2 polyethylene (PEG) QDs and elemental co-localization analysis of two elements present in the QDs (sulfur and cadmium) was performed on putative intracellular QDs with electron spectroscopic imaging (ESI). Both elements were shown on a single particle level and QDs were confirmed to be located inside intracellular vesicles. Nevertheless, ESI analysis showed that not all nano-sized structures, initially identified as QDs, were confirmed. This observation emphasizes the necessity to perform elemental analysis when investigating intracellular NP localization using TEM.

Investigating pharmacology in vivo using magnetic resonance and optical imaging.

The better and earlier a disease can be diagnosed and characterized, the greater the chance of being able to intervene in this process with a chemical entity. This is the rationale for the use of in vivo imaging techniques in the drug discovery and development process. In this article we address the value of two imaging modalities in this area, i.e. magnetic resonance imaging (MRI) and optical imaging. The multiparametric nature of MRI enables anatomical, functional, metabolic and, to a certain extent, also cellular and target-related information to be obtained noninvasively at high spatial resolution. This favours characterization of a disease state and the corresponding drug intervention. The noninvasiveness of MRI strengthens the link between preclinical and clinical pharmaceutical research. The high sensitivity of optical techniques enables molecular information to be obtained in vivo. Within pharmacological research, the main applications of optical techniques relate to the early drug discovery process and acquisition of target-related information. However, potential clinical applications of optical imaging are also emerging. The complementary character of both imaging modalities renders them useful in various portions of the drug discovery process, from early target selection and validation to clinical studies.

Polyphosphate at the Streptomyces lividans cytoplasmic membrane is enhanced in the presence of the potassium channel KcsA.

The distribution of polyphosphate (polyP) within the cytoplasmic membrane of Streptomyces lividans hyphae or protoplasts has been determined at high spatial resolution by elemental mapping using energy-filtered electron microscopy (EFTEM). The results revealed that polyP was best traceable after its interaction with lead ions followed by their precipitation as lead sulphide. Concomitant studies of the S.lividans wildtype (WT) strain and its co-embedded mutant DeltaK (lacking a functional kcsA gene) were conducted by labelling as the surface matrix of either one was labelled by cationic colloidal thorium dioxide. Within the WT strain, additional polyP was found to accumulate distinctly at the inner face of the cytoplasmic membrane. After removal of the cell wall (within protoplasts), the polyP-derived lead-sulphide (PbS) precipitate formed clusters of fibrillar material extending up to 50 nm into the cytoplasm. This feature was absent in the DeltaK mutant strain. Together the results revealed that the presence of the KcsA channel and the structured polyP coincide.

Towards high-resolution three-dimensional imaging of native mammalian tissue: electron tomography of frozen-hydrated rat liver sections.

Cryo-electron tomography of frozen-hydrated specimens holds considerable promise for high-resolution three-dimensional imaging of organelles and macromolecular complexes in their native cellular environment. While the technique has been successfully used with small, plunge-frozen cells and organelles, application to bulk mammalian tissue has proven to be difficult. We report progress with cryo-electron tomography of frozen-hydrated sections of rat liver prepared by high-pressure freezing and cryo-ultramicrotomy. Improvements include identification of suitable grids for mounting sections for tomography, reduction of surface artifacts on the sections, improved image quality by the use of energy filtering, and more rapid tissue excision using a biopsy needle. Tomographic reconstructions of frozen-hydrated liver sections reveal the native structure of such cellular components as mitochondria, endoplasmic reticulum, and ribosomes, without the selective attenuation or enhancement of ultrastructural details associated with the osmication and post-staining used with freeze-substitution.

Near-infrared spectroscopy and imaging: basic principles and pharmaceutical applications.

Near-infrared (NIR) spectroscopy and imaging are fast and nondestructive analytical techniques that provide chemical and physical information of virtually any matrix. In combination with multivariate data analysis these two methods open many interesting perspectives for both qualitative and quantitative analysis. This review focuses on recent pharmaceutical NIR applications and covers (1) basic principles of NIR techniques including chemometric data processing, (2) regulatory issues, (3) raw material identification and qualification, (4) direct analysis of intact solid dosage forms, and (5) process monitoring and process control.

Large-scale homogeneous molecular templates for femtosecond time-resolved studies of the guest-host interaction.

Self-assembled monolayer films based on iodobenzoyloxy-functionalized resorc[4]arenes were prepared on gold substrates to serve as model systems for future time-resolved studies of molecular recognition, a mechanism of outstanding importance in bioorganic systems. The film properties were tested using X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM) and imaging ellipsometry. An apparatus for time-resolved electron spectroscopy utilizing femtosecond soft X-ray pulses is capable of detecting iodine core-level photolines and the photoinduced dissociation after ultraviolet illumination. The developed technique holds promise for tracking the temporal evolution of chemical shifts of atomic markers as local probes for the dynamics of the guest-host interaction.